Mast cells are effector and regulatory cells involved in the pathogenesis of allergic inflammation, generally through the activation of the high affinity IgE receptor (Fc-epsilon-RI). Binding of an antigen to IgE/Fc-epsilon-RI complexes initiates intracellular signaling events that lead to the release of vasoactive and inflammatory cytokines. These mast cell-derived products mediate immediate and delayed allergic reactions. The signaling pathways linking Fc-epsilon-RI to human mast cell activation are, however, incompletely understood. In addition to the IgE receptor, mast cells express a variety of cell surface receptors and when activated, these receptors can modify signaling and responses to an antigen. A deeper understanding of IgE receptor-mediated signaling pathways and of those by other receptors that modulate the responses to the IgE receptor stimulation can give clues on treatment options for human allergic disease. During FY 2016, we found that DJ-1, an evolutionary conserved protein reported to protect cells against oxidative damage, is abundantly expressed in mast cell lines and that the serum levels of DJ-1 are altered in patients with atopic dermatitis and in patients with systemic mastocytosis. Our further studies in FY2017,on the role of DJ-1 in human mast cells revealed an unsuspected role for DJ-1 in the early signaling of Fc-epsilon-RI and in the consequent release of mast cell mediators. When the IgE/Fc-epsilon-RI complex encounters an antigen, activation of the Src kinase Lyn is the initiating step of a series of phosphorylation events driving mast cell responses. We demonstrated that early after activation of Fc-epsilon-RI, DJ-1 translocates to specialized lipid domains or lipid rafts, where it directly interacts with Lyn. This interaction was critical for full Lyn activation, downstream signaling pathways and for mast cell degranulation, cytokine and prostanoid release in response to antigen. Such interaction, which occurred only with non-oxidized DJ-1, facilitated the active conformation of Lyn independently of the antioxidant function of DJ-1. In FY2018, we are further characterizing the DJ-1/Lyn interaction which may open new avenues of therapeutic modalities in mast cell-derived diseases. Activation of Fc-epsilon-RI on mast cells leads to the generation and secretion of the sphingolipid mediator, sphingosine-1-phosphate (S1P) which is able, in turn, to transactivate its receptors on mast cells. Previous reports have identified the expression of two of the five receptors for S1P on mast cells, S1P1 and S1P2, with functions in FcRI-mediated chemotaxis and degranulation, respectively. In FY2017 and continuing into FY2018, we showed that cultured mouse and human mast cells also express abundant message for S1P4. Although deletion of S1pr4 did not affect the differentiation or proliferation of bone marrow progenitors into mast cells, or IgE-mediated mast cell degranulation, we uncovered a role for S1P4 in the negative regulation of degranulation of innate mast cells in response to co-stimulation with IgE/Ag and IL-33. In FY 2018, we began to extend these findings in human mast cells. IL-33 is thus emerging as a critical player orchestrating allergic inflammation through innate immune cells, including mast cells. Increases in IL-33 in the epithelia are caused by barrier defects, microbiome alterations, irritants, allergens and other substances. IL-33 induces cytokine production in mast cells and promotes degranulation-associated responses leading to exacerbated sensitization to food or airway allergens. As mast cells can also downregulate IL-33 actions in other models of inflammation, a better understanding of the mechanisms and circumstances under which S1P4 modulates IL-33 actions in mast cells may be beneficial for learning how to tamper certain allergic conditions. Sphingosine-1-phosphate (S1P) is a pleiotropic lipid mediator that has important roles in proliferation and survival in many cell types. The two isoforms of sphingosine kinase (SPHK1 and SPHK2) that generate S1P can be activated by Fc-epsilon-RI, KIT and IL-3 stimulation in mast cells. Since excessive accumulation of mast cells is observed in proliferative mast cell disorders, we questioned whether SPHK-S1P pathways may regulate mast cell growth. Our studies completed in FY2017 continuing into FY2018 revealed that both SPHK1 and SPHK2 are important regulators of the proliferation and survival of normal and neoplastic mast cells. Moreover, neoplastic mast cells with oncogenic KIT, a receptor that promotes mast cell differentiation and growth, are particularly dependent on the SPHK1 isoform. Inhibition of SPHK1 induced the activation of the DNA damage cascade and resulted in G2/M arrest and cell death. The results suggest a role for SPHK1 in oncogenic pathways by KIT, preventing or repairing double-strand DNA breaks during oncogene-induced replication stress. In addition, the findings suggest the usage of SPHK inhibitors may be useful alternatives to tyrosine kinase inhibitors in the treatment of aggressive systemic mastocytosis. Studies on mast cell responses and mechanisms of activation often rely on cultured cells. Human mast cells can be differentiated in vitro from blood progenitors using specific protocols that tend to be time-consuming and costly. Furthermore, individual blood withdrawals from human subjects are in the range of 100 ml and this tends to limit the amount of cells that can be obtained from a single subject's visit. In FY2017 continuing into FY2018, we developed and validated a modified method for culturing human mast cells that uses 30 to 50 times less media and consumables and is thus significantly more cost effective and also less labor intensive. We found that this method can be successfully applied, with similar yields, for differentiation of mast cells from cryopreserved lymphopheresis cells, which has the advantage that can be used over time from a single visit, facilitating not only cost but also accessibility to samples. These methodological improvements will significantly facilitate human mast cell research.